Under normal circumstances, the endothelium regulates vascular homeostasis via its influence on vasomotor tone, platelet function, leukocyte trafficking, vascular remodeling, and angiogenesis. Many aspects of these functions are regulated, in part, by the endothelial production of nitric oxide (NO7). In patients with vascular diseases such as hypertension and atherosclerosis, NO bioactivity is impaired predisposing these patients to vascular events including myocardial infarction and stroke. Impaired NO bioactivity has been linked to excess vascular production of reactive oxygen species (ROS), particularly superoxide (7O2-), that rapidly reacts with NO7 to quench its bioactivity. The NADPH oxidase (Nox) family of enzymes plays a prominent role in pathologic ROS production that limits NO7 bioactivity. In this application, however, we present data that NADPH oxidase isoform 4 (Nox4) is an endothelial ROS source that paradoxically promotes normal NO7 bioactivity. Our data indicate that intracellular ROS produce contextual responses based upon the site of ROS production and the type of ROS produced. Our findings will radically change current paradigms involving NO7 and ROS in the vasculature and will have broad implications beyond vascular disease. The central hypothesis of this proposal, therefore, is that Nox4 is an important determinant of endothelial cell phenotype based upon contextual ROS signaling that contributes to normal vascular homeostasis. The objective of this proposal is to identify determinants of physiologic Nox4 signaling in the endothelium and the underlying molecular mechanisms involved in this process. In order to achieve this objective, we will first determine the molecular mechanisms for regulation of Nox4 catalytic activity in the endothelium. These studies will involve endothelial and COS-7 cells to determine the specific domains of Nox4 that dictate its intracellular localization and catalytic activity. Then we will examine how certain receptor ligands, such as EGF, and VEGF modulate Nox4 catalytic activity and intracellular localization. Studies will also be performed in mouse aortic endothelial cells (MAECs) from mice that either overexpress or lack Nox4 in the endothelium. We will then move on to determine the molecular mechanisms responsible for Nox4-mediated modulation of eNOS activity. Genetic manipulation of Nox4 levels in the endothelium will help us determine the implications for NO7 bioactivity and eNOS catalysis. We will then probe the involvement of known Nox4 targets such as Akt, PTP1B, and SOD1. Our data implicate Nox4 in VEGF signaling, prompting us to define the precise mechanisms involved. These studies will be used to set the stage for determining the implications of Nox4 on endothelial cell phenotype in cell culture such as proliferation, migration, and angiogenesis. Finally, we will determine the implications of Nox4 on endothelial cell phenotype and vascular disease in vivo using mice that either overexpress or lack Nox4 in the endothelium. These animals will be used to probe endothelial Nox4 on vascular NO7 bioactivity and angiogenesis. We expect these experiments to provide us with a solid working knowledge of how Nox4 contributes to the control of endothelial phenotype and how this translates into homeostatic responses in vivo. With this information in hand, we should have the requisite insight to design new tools directed at modulating vascular phenotype with an eye toward the treatment of vascular disease. PUBLIC HEALTH RELEVANCE: The endothelium is the lining of blood vessels and its behavior is an important control point for blood vessels. We know from experience that blood vessels in people at risk for atherosclerosis do not work normally. In fact, those individuals with the worst function in their blood vessels are at the highest risk for heart attack. Current dogma suggests that the production of free radicals in blood vessels is responsible for some of the abnormal function of blood vessels in the setting of disease. However, in this proposal, we provide evidence that free radicals produced in the blood vessels are actually part of normal function. We have found a particular enzyme, known as Nox4 that produces radicals in a manner that helps the normal function of blood vessels. This proposal is designed to determine how this enzyme, Nox4, produces more normal function.